To be able to establish, maintain, and exit from the pluripotent state, embryonic stem cells require dynamic and precise coordination of their molecular machinery. Significant headway has been made in elucidating the epigenetic, transcriptional, and non-coding RNA-driven post-transcriptional mechanisms that govern this unique state, but far less is known about the RNA binding proteins (RBPs) that directly enable the post- transcriptional and translational regulation of gene expression, despite evidence of their dominant role in shaping the molecular landscape of ESCs. The overall goal of the proposed project is to define the functions of a specific RBP in pluripotency. To this end, I conducted an RNAi screen of 356 putative RBPs in an assay tracking the differentiation of ESCs to epiblast-like cells (EpiLCs), a progression that represents the first cell fate decision made in the embryo proper. Interleukin enhancer-binding factor 2 (Ilf2) was identified as a candidate RBP whose knockdown promoted differentiation to the EpiLC state. Ilf2 is a remarkable protein, because it appears to participate in many different molecular processes extending across transcription, splicing, translation, and miRNA biogenesis. While there is circumstantial evidence that Ilf2 is involved in pluripotency, it is unknown where in the spokes of the pluripotency network Ilf2 is situated. I hypothesize that Ilf2 controls the expression pattern f a core set of ESC-specific target genes through simultaneous participation in multiple regulatory layers, and the combination of these activities promotes the pluripotent state. Ilf2-/- and wild-type (wt) ESCs will be compared to determine the validity of this hypothesis. Specifically, Aim 1 will characterize the effects of Ilf2 loss on ability of ESCs to self-renew and differentiate. Aim will determine whether Ilf2 directly affects transcription, transcript stability, and translation i ESCs. Finally, Aim 3 will assess the role of Ilf2 in the miRNA regulation of ESCs. Addressing these aims will not only increase appreciation of the scope of RBP involvement in pluripotency but also enable the more informed use of ESCs as an in vitro platform for disease modeling, drug discovery, and tissue regeneration. This research project is also designed to promote my development as a future physician-scientist and educator capable of leading an independent research lab. The multidisciplinary skills needed to achieve the proposed aims will provide me solid training in both wet lab techniques and computational methods. My training will also include coursework in developmental biology, bioinformatics, ethics, and medicine. Additionally, I will gain experience in pedagogy by mentoring high school, undergraduate, graduate, and medical students.